Selenium (Se) is an essential micronutrient and is also used in various industrial processes. However, Se also exhibits a low toxicity threshold and therefore presents a significant risk to human kind when released into the environment. The gap between Se deficiency (< 40 µg•day−1) and acute Se poisoning (> 400 µg•day−1) for humans is rather narrow. In addition, detrimental effects to the health of humans and other biota can arise from radioactive Se isotopes. Namely, 79Se is of concern, as it is one of the fission products originating from nuclear power production. The toxicity of selenium not only depends on its concentration but also on its speciation. This of course applies to both stable and radioactive isotopes. Microorganisms play a key role in determining and altering the speciation of Se in the selenium geochemical cycle. The naturally released selenium oxyanions (selenite (SeIVO32−) and selenate (SeVIO42−)) can be microbially reduced to differently shaped biogenic elemental selenium (BioSe, Se(0)) nanomaterials - BioSe-Nanospheres and BioSe-Nanorods. Even after more than 30 years of elaborated research on selenium, the impact of the microbial biota on the shape change of these BioSe-Nanomaterials lacks a fundamental understanding. Furthermore, due to the various species of microorganisms having different metabolisms, a detailed investigation of representative organism is required to predict the fate of selenium in the environment and engineered systems. Thus, the motivation behind this Ph.D. work was to study the effect of selected microorganisms (based on their high resilience, application in wastewater treatment processes, and capability to reduce selenium oxyanions) on the properties and fate of the produced biogenic elemental selenium nanomaterials. Namely, this meant deciphering the role of selenium oxyanion reduction mechanism on the localisation (intracellular or extracellular) of the microbially produced biogenic elemental selenium nanoparticles. This understanding is important as the localisation defines the release of the selenium nanoparticles in the environment and hence its potential pathway into the food chain. Further, the role of the microorganisms (pure culture and mixed culture) on the composition and stability of the corona (organic layer) on the BioSe-Nanomaterials was studied as properties of the corona can affect the stability and hence the localization of the nanomaterials. Moreover, the effect of the microbial environment on the shape establishment and stability, as well as on the fate of the produced biogenic elemental selenium nanomaterials was also investigated. Eventually, the obtained results narrow the identified knowledge gap and improve the understanding of the fate of selenium in the environment. In the first part of this Ph.D. thesis, the bacterial strain Bacillus safensis JG-B5T was chosen to study the influence of microbes on the fate of Se in the environment due to its occurrence in uranium mining sites where selenium is also found. First, this bacterium has been analysed by genome sequencing and its genomic data were deposited at the NCBI database. With the obtained results, the bacterial strain was classified in the corresponding phylogenetic tree. Furthermore, this Ph.D. work revealed that B. safensis JG-B5T is an obligate aerobic microorganism with the ability to reduce SeO32− to elemental selenium (Se(0)) in the form of red BioSe-Nanospheres. A reduction of SeO42− has not been observed. Two-chamber reactor experiments revealed that direct contact between SeO32− and the bacterial cells was necessary to start the reduction. In addition, microscopic investigations identified changes in the bacterial cell morphologies induced by toxic stress effects of SeO32−. Only extracellular production of BioSe-Nanospheres was observed using STEM equipped with a HAADF detector. The produced BioSe-Nanospheres were characterized by Raman spectroscopy as being amorphous Se. Furthermore, a stabilizing corona containing proteins and EPS, which caps the BioSe-Nanospheres, has been identified by FT-IR spectroscopy. The detailed composition of this corona has been further studied using proteomics analysis. The combination of two-chamber reactor experiments, genome analysis and the identified corona proteins indicated that the selenite reduction process of B. safensis JG-B5T was primarily mediated through membrane-associated proteins, like succinate dehydrogenase. Thus, a detailed molecular mechanism of the microbial reduction of SeO32− to BioSe-Nanospheres by the bacterial strain B. safensis JG-B5T has been proposed within this work. Besides these investigations on the formation of BioSe-Nanospheres, ζ-potential measurements have shown a low colloidal stability of the produced BioSe-Nanospheres. Thus, B. safensis JG-B5T is an attractive candidate in selenite wastewater treatment as it provides easy ways of recovering Se while maintaining low Se discharge. These investigations motivated us to study the general role of the microbial origin and microbial environment of the discharged nanomaterials in their shape change from BioSe-Nanospheres to BioSe-Nanorods. This constitutes the second part of this Ph.D. thesis. Thus, two different known microbial BioSe-Nanospheres producers by means of selenite reduction were used, namely the bacterial strain Escherichia coli K-12 and the microbial mix culture of anaerobic granular sludge. It was shown with Raman spectroscopy and SEM imaging that the BioSe-Nanospheres produced by E. coli K-12 remain amorphous and spherical when exposed to thermophilic conditions (up to one year), whereas those obtained by anaerobic granular sludge transform to trigonal BioSe-Nanorods. ζ-potential measurements identified a decrease of the colloidal stability of the transformed BioSe-Nanorods of anaerobic granular sludge compared to the still spherical BioSe-Nanospheres of E. coli K-12. As the shape of these BioSe-Nanospheres is stabilized by their corona, detailed investigations were performed to derive key factors affecting its shape change. CheSeNMs capped with different amount of BSA were produced and incubated to evaluate the quantitative effect of the amount of proteins in the corona on the shape stability of BioSe-Nanomaterials. This experiment implied that the larger quantity of proteins present in the corona of the BioSe-Nanospheres provide better shape stability. Indeed, the BioSe-Nanospheres produced by E. coli K-12 have 5.5 times more protein than those produced by anaerobic granular sludge. To gain deeper insight into their structural properties, proteomics analysis identified the surface proteins of the BioSe-Nanomaterials. The proteomics analysis also showed that the corona of BioSe-Nanospheres produced by E. coli K-12 consists of 1009 different proteins compared to only 173 on those produced by anaerobic granular sludge. The possible difference in the interaction of the corona proteins and selenium was elucidated using density functional theory calculations. The calculations suggest the possibility of the S-Se bond formation between Se atom and sulphur of the cysteine and methionine residues of the corona proteins. Furthermore, as representative for the microbial environment the bacterial strain B. safensis JG-B5T was used to mimic the role of microorganisms living in the vicinity of the discharged nanoparticles. The bacterial strain was incubated with purified BioSe-Nanospheres produced by E. coli K-12 at mesophilic conditions. Raman spectroscopy and SEM imaging showed that in contrast to the thermophilic incubation, the BioSe-Nanospheres transformed to BioSe-Nanorods in the presence of B. safensis JG-B5T. Proteomics analysis identified that the protein corona of BioSe-Nanospheres produced by E. coli K-12 was degraded by extracellular peptidases secreted upon co-incubation with B. safensis JG-B5T bacteria, which led to their transformation to BioSe-Nanorods. All the above findings show, how microorganisms fundamentally impact the speciation, colloidal stability, and shape of selenium. These, consequently, affect their flow coefficients or partition factors in the environment and therefore their fate. This work consequently demonstrates that the shape of the BioSe-Nanomaterials depends on both, their microbial origin and their microbial surrounding. Especially, the dynamic changes induced by this microbial environment on the shape of already formed BioSe-Nanospheres after their discharge are to be further explored. This increases the complexity in determining the risk assessment of Se and probably other redox active elements, which needs to be re-evaluated and improved by including microbial criteria for better accuracy. Based on the presented investigations, further studies regarding the detailed application and expansion to other bacterial strains will continuously widen the understanding of the behaviour of Se in the environment and engineered systems.
| Identifer | oai:union.ndltd.org:DRESDEN/oai:qucosa:de:qucosa:86556 |
| Date | 25 July 2023 |
| Creators | Fischer, Sarah |
| Contributors | Stumpf, Thorsten, Henle, Thomas, Jain, Rohan, Jordan, Norbert, Technische Universität Dresden |
| Source Sets | Hochschulschriftenserver (HSSS) der SLUB Dresden |
| Language | English |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, doc-type:doctoralThesis, info:eu-repo/semantics/doctoralThesis, doc-type:Text |
| Rights | info:eu-repo/semantics/openAccess |
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